The ability to synthesize polynucleotide fragments having a desired nucleotide sequence is a useful tool in both research and applied molecular biology. Short synthetic polynucleotides, or oligonucleotides, are useful as adaptors or linkers in joining longer DNA segments, and as hybridization probes and DNA synthesis primers. Longer polynucleotides can be constructed from shorter segments having overlapping cohesive ends and used as structural genes, regulatory regions such as promoters, terminators, operators, and the like. It is thus of great interest to provide convenient automatic techniques for producing synthetic DNA fragments with high yields in a relatively short time.
As the understanding of the function, structure, and chemical makeup of nucleotide sequences, such as DNA, has evolved, so too has the awareness of the practicalities and feasibilities of genetic engineering. These engineering efforts, however, require a complete understanding of the chemical and biological reactions in cells. One of these reactions is the postreplicative modification of newly synthesized DNA by the selective methylation of certain cytosine residues which is performed enzymatically by a specific DNA methylase. An understanding of the factors governing the formation of specific methylation patterns in eucaryotic DNA is very important if we are to understand the mechanisms of gene expression.
Basic to such genetic engineering efforts is the synthesis of desired nucleotide chains from single mononucleotides. In this regard, electromechanical apparatus has been developed for synthesizing desired oligonucleotide sequences via the sequential linking of desired bases to a starting nucleotide.
At present, a variety of approaches for polynucleotide synthesis are available. These approaches can be characterized based on several criteria. First, the synthesis is usually carried out either on a solid-phase substrate or in solution. Solid-phase synthesis relies on sequential addition of mononucleotides to a growing chain attached at one end to the substrate. The solid phase permits easy separation of the reactants, but the method requires excess quantities of reactants and usually provides only small quantities (less than 1 mg) of the desired sequence. Solution phase synthesis, while it requires lesser amounts of the expensive reagents and can provide larger quantities of the product sequence, requires isolation and purification of the intermediate product after every addition. Virtually all automated polynucleotide systems rely on solid phase synthesis.
There are presently two synthesis chemistries in widespread used for automated polynucleotide synthesis. The triester method, as described by Catlin and Cramer J. Org. Chem. 38: 245-250 (1973) and Itakura et al., Can. J. Chem. 51: 3649-3651 (1973) which relies on the addition of suitable blocked phosphate-triester intermediates which are generally inexpensive and stable. The phosphite-triester method, as described by Letsinger and Lunsford in J. Am. Chem. Soc. 98:3655 (1975) is somewhat more complex, but generally provides higher yields than the phosphate triester method. The utility of the phosphite-triester method was greatly improved by the use of N,N-dialkylamino phosphites (amidites) which are more stable than the phosphor-chlorodite intermediates initially employed. While the phosphite-triester method is often favored because of the greater yield at each nucleotide addition, the phosphate-triester method is also suitable for automated polynucleotide synthesis.
Among the reactor systems that can be used in synthesizing polynucleotides are solid-phase reactor systems which use either a tight bed column, a loose bed column, or a batch reactor. The tight bed column is tightly packed with the solid-phase support and the reactants are introduced either in a single pass or by a recirculating stream.
Loose bed columns have been introduced to alleviate these problems partially. By slowly passing the reactant through the column, higher mass transfer rates are achieved and utilization of the expensive reactants is improved. Also, channelling is reduced, since the solid phase packing will shift to equalize the flow profile therethrough.
In a batch reactor, the support matrix is held in an enclosed vessel. Reactants are introduced and the vessel contents agitated, typically by bubbling an inert gas through the liquid in the reactor. While such a system can provide very efficient utilization of the reactants by increasing the retention time in the reactor, relatively large volumes of the reactant and solvent are necessary to fill the reactor.
Urdea et al., in U.S. Pat. No. 4,517,338, disclose a method and system for sequential modification of a linear polymeric molecule attached to a dispersed solid phase support by adding individual nucleotides in a predetermined order to a nucleotide chain. The dispersed solid phase is retained within a reactor zone which is provided with access ports for the introduction and removal of reagents. Reagents are selectively delivered to the reactor zone through at least one of the access ports by a reagent manifold.
Another apparatus for programmably synthesizing selected nucleotide sequences is described in Zelinka et al., U.S. Pat. No. 4,598,049.
The well known instability of the triazine ring of 5-azacytosine deoxyribonucleoside makes it unsuitable for use as a building block in the aforementioned automated DNA syntheses. Despite this drawback, interest in the synthesis of single and double stranded DNA fragments containing 5-azacytosine residues constitutes an important goal in the understanding of the mechanism of action of this drug. DNA incorporation of 5-azacytosine in living cells has been associated with inhibition of DNA methylase activity and consequent gene activation.
Because of the well established relationship that exists between the DNA incorporation of 5-azacytosine residues and gene activation, it would be o useful to develop a methodology for the synthesis of oliqonucleotide fragments which contain this unnatural base. These modified oligonucleotides, which would contain 5,6-dihydro-5-azacytosine and 5-azacytosine residues at specific sites, could serve as tools for elucidating the mechanism of selective gene activation and the relationship that exists between the presence of these triazine bases and inhibition of DNA methylation. A direct incorporation of the phosphoramidite of 2'-deoxy-5-aza-cytidine in DNA synthesis would results in failure, since the 2'-deoxy-5-aza-cytidine is extremely sensitive to acid or alkaline conditions.